PH20 polypeptide variants, formulations and uses thereof
Modified PH20 hyaluronidase polypeptides, including modified polypeptides that exhibit increased stability and/or increased activity, are provided. Also provided are compositions and formulations and uses thereof.
1. A modified PH20 polypeptide, comprising one or more amino acid modifications in an unmodified PH20 polypeptide, wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7 and 32-66;
amino acid modifications are selected from the group consisting of amino acid replacements(s), deletion(s), and/or insertion(s);
the modified PH20 polypeptide comprises an amino acid replacement at a position corresponding to residue 324, with reference to amino acid positions set forth in SEQ ID NO: 3;
the replacement at the position corresponding to residue 324 is selected from the group consisting of A, D, H, M, N, R and S;
corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide having the amino acid sequence of SEQ ID NO: 3; and
the modified PH20 polypeptide has at least 91% sequence identity to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7 and 32-66.
2. The modified PH20 polypeptide of claim 1 , wherein the modified PH20 polypeptide has at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, and 32-66.
3. The modified PH20 polypeptide of claim 1 that has increased resistance to or stability in denaturing conditions compared to an unmodified PH20 polypeptide that does not contain the amino acid modification(s).
4. The modified PH20 polypeptide of claim 1 that exhibits increased hyaluronidase activity compared to the unmodified PH20 polypeptide not containing the amino acid replacement at position 324.
5. The modified PH20 polypeptide of claim 1 that is a soluble PH20 polypeptide.
6. The modified PH20 polypeptide of claim 1 , wherein the replacement at the position corresponding to residue 324 is D.
7. The modified PH20 polypeptide of claim 1 , wherein the replacement at the position corresponding to residue 324 is N or R.
8. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66.
9. The modified PH20 polypeptide of claim 7 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66.
10. The modified PH20 polypeptide of claim 6 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from among the group consisting of SEQ ID NO: 3 and 32-66.
11. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.
12. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.
13. The modified PH20 polypeptide of claim 6 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.
14. The modified PH20 polypeptide of claim 6 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.
15. The modified PH20 polypeptide of claim 1 , comprising a sequence of amino acids that exhibits at least 91% sequence identity to the sequence of amino acids selected from the group consisting of SEQ ID NO: 3, and 32-66 and that contains an amino acid replacement D at the residue corresponding to residue 324 with reference to SEQ ID NO: 3.
16. The modified PH20 polypeptide of claim 1 that is C-terminally truncated, whereby the polypeptide is soluble.
17. The modified PH20 polypeptide of claim 1 that comprises one or more post-translational modifications of the polypeptide selected from the group consisting of glycosylation, sialylation, albumination, farnesylation, carboxylation, hydroxylation, and phosphorylation.
18. The modified PH20 polypeptide of claim 17 , wherein the post-translational modification is glycosylation.
19. The modified PH20 polypeptide of claim 18 , wherein the polypeptide is a glycoprotein that comprises an N-acetylglucosamine moiety linked to each of at least three asparagine (N) residues.
20. The modified PH20 polypeptide of claim 1 that is conjugated to a polymer.
21. The modified PH20 polypeptide of claim 20 , wherein the polymer is dextran or polyethylene glycol (PEG).
22. The modified PH20 polypeptide of claim 1 , further comprising a heterologous signal sequence, wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66.
23. A chimeric polypeptide, comprising the modified PH20 polypeptide of claim 1 .
24. A pharmaceutical composition, comprising the modified PH20 polypeptide of claim 1 .
25. The modified PH20 polypeptide of claim 6 , wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32; and
the amino acid sequence of the modified PH20 polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:32.
26. The modified PH20 polypeptide of claim 6 , wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35; and
the amino acid sequence of the modified PH20 polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:35.
27. The pharmaceutical composition of claim 24 , further comprising a therapeutically active agent formulated in the same composition or in a separate composition.
28. The pharmaceutical composition of claim 27 , wherein the therapeutically active agent is a polypeptide, a protein, a nucleic acid, a drug, a small molecule, or an organic molecule.
29. The pharmaceutical composition of claim 27 , wherein the therapeutically active agent is selected from the group consisting of a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, an antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, a urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, and a sleep inducer.
30. The pharmaceutical composition of claim 27 , wherein the therapeutically active agent is an antibody.
31. A method for treating a hyaluronan-associated disease or condition, comprising administering to a subject a modified PH20 polypeptide of claim 1 .
32. The method of claim 31 , wherein the hyaluronan-associated disease or condition is an inflammatory disease or a tumor or cancer.
33. The method of claim 32 , wherein the hyaluronan-associated disease or condition is a solid tumor.
34. The modified PH20 polypeptide of claim 1 that is further modified by conjugation to a moiety selected from the group consisting of a multimerization domain, a toxin, a detectable label, and a drug.
35. The modified PH20 polypeptide of claim 34 , wherein the modified PH20 polypeptide is conjugated to a multimerization domain that is an Fc domain.